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In Vivo Imaging System in Gene Therapy

In Vivo Imaging System in Gene Therapy

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Published by erhan6936
A novel in vivo imaging system develoed for following cancer or other disease
A novel in vivo imaging system develoed for following cancer or other disease

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Categories:Types, Research, Science
Published by: erhan6936 on Mar 29, 2009
Copyright:Attribution Non-commercial

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01/30/2013

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Xenogen Corporation has developeda technology known as biophotonicimaging (3, 5, 26) which allows bio-logical processes, including geneexpression that is both temporal andspatially defined (e.g., occurring indefined tissues and organs within theanimal), to be monitored in live ani-mals in real-time. Genes encoding spe-cific luciferase proteins are engineeredinto cells (e.g., bacterial pathogensand cancer cell lines) and animals(transgenic mice) to enable them toproduce light that can be visualizedthrough the tissues of a live animalusing specialized imaging equipment and software designed and built by the company. Todate, Xenogen’s technology has been used predominantly to facilitate drug discovery inareas such as infectious disease (9, 10, 14, 27), oncology (4, 7, 25), inflammation and toxi-cology (6, 36, 37). Recently, this technology has also been used for the assessment of thecapability of RNAi molecules to regulate gene expression in live animals (20, 21, 32),enabling a researcher to more rapidly assess whether an RNAi is being delivered to thetarget tissue to effectively reduce translation of a specific mRNA. This overview gives ascientific approach on how biophotonic imaging can be used to facilitate research anddevelopment of RNAi in live animals, and provides an insight into how small RNAimolecules might be better developed as human therapeutics.
Overview of Xenogen Technology
Bioluminescence is a biological process by which certain organisms can generate lightthrough an enzyme-mediated reaction. Firefly, glowworm and certain bacteria (commonlyassociated with fish and squid) are probably the most familiar examples of this phenomenon,all producing visible light. The proteins involved in both firefly and bacterial biolumines-cence have been identified and the genes that encode them have been cloned. In both cases,the proteins responsible for bioluminescence are called luciferases. These enzymes generatebioluminescence via a biological reaction in which oxygen and a luciferin substrate react inthe presence of a cellular energy source (e.g., ATP) to produce photons of light.The application of these bioluminescent systems to monitor gene expression in cells is nowroutine in molecular and cellular biology. Typically, the luciferase gene(s) is cloned adjacentto the region of a gene controlling expression (the promoter), such that the luciferase is pro-duced in a fashion similar to that of the native protein. Bioluminescence can be monitored
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HEETRNAi In Vivo Applications
Living Image
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Software
IVIS
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Imaging System
In vivo biophotonic imaging is offered with the XenogenIVIS
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Imaging System. Living Image
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software controlsthe imaging process, and analyzes and archives data.
 
from cells containing theseluciferases using a light sensi-tive detector, such as a lumi-nometer. Xenogen uses theabove approach, but appliesit to monitoring real-timeluciferase expression in livinganimals; a technique termed“in vivo biophotonic imag-ing.” In the same way thatbioluminescent light is trans-mitted from cells within thefirefly tail, or bacterial cellswithin a symbiont (e.g., flashlight fish), so light emitted from bioluminescently engineeredcells (e.g., pathogenic bacteria, cancer cells, or transgenic tissue) placed or generated withina small animal (e.g., a mouse or rat) can be detected at the surface (26). Animal tissue willallow light passage to some degree (imagine a flashlight held behind a hand and seeing thered light shining through), and with a suitably sensitive detector (e.g., CCD camera) andimage processing software, low levels of light emitted by bioluminescent cells within ananimal can be detected, transformed into graphic displays and analyzed. Xenogen hasperfected this technique by designing and building its own imaging system, the IVIS
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Imaging System, as well as Living Image
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analysis software.Typically, the bioluminescent light generated by genetically engineered cells can penetrate1–2 cm of tissue making mice ideal subjects to monitor such activity. The location andnumber of such cells can then be tracked in the live animal. Moreover, the same animalmay be imaged multiple times, so allowing the expansion or regression of the disease tobe followed (e.g., during infectious disease or oncology studies).Biophotonic imaging isunique in that it can beapplied to monitor virtuallyany biological process inreal-time in a live animal,whether that process be theinduction of a particularcytokine by the host (e.g.,mouse IL-6) in response to aninvading pathogen, or a viru-lence factor induced in apathogen (e.g., bacterial hemolysin) in response to its invasion of a host. Furthermore,because different luciferases often use different substrates and emit light at differentwavelengths, as in the case of firefly and bacterial luciferase, it is possible to monitortwo biological events in the same animal at the same time. Thus, in the above example
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Xenogen In Vivo RNAi Applications
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In vivo biophotonic imaging incorporates bioluminescently engineeredBioware
cells or microorganisms, as well as LPTA
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animal modelsgenetically engineered to express firefly luciferase.
Tag Cell or BacteriaTag GeneIVIS
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Imaging System• Digitize• Quantify• Archive
 
it should be possible to monitor both the induction of the hemolysin in the bacteria as itinfects the host, and the host’s response to this bacterium with regard to its IL-6 induction.In addition to a large number of infectious disease (9, 10, 14) and oncology (4, 7, 25) ani-mal models that have been developed at Xenogen, an extensive program has also beenestablished for the generation of transgenic animals expressing firefly luciferase, designatedas LPTA
animal models, under the control of different inducible promoters [e.g., induciblenitric oxide synthase promoter, VEGFR2 promoter, rat insulin promoter, heme oxygenasepromoter (6, 37) and bone morphogenesis protein 4 promoter (36)]. These LPTA
®
animalmodels allow the effects of a particular compound (chemical or biological) to be visualizedin the whole animal as that compound is absorbed and metabolized by the differenttissues/organs of that animal. Thus, multiple data points can be collected over time andfrom different regions (tissues/organs) within the same animal.
Background on RNAi Research
Since the first successful report of the use of small interfering RNA (siRNA) to silence geneexpression in mammalian cells (8), a flood of papers reporting the use of RNA interference(RNAi) to elucidate mammalian gene function has followed. Delivery of synthetic siRNAsto mammalian cells in culture can be achieved using lipophilic agents or electroporation.Alternatively, interfering RNAs can be expressed from a plasmid harbored by the cells ofinterest, in which pairs of short complimentary RNA molecules (17, 23, 35), or a singleinverted small hairpin RNA (shRNA) are stably expressed and used for RNAi gene silencing(1, 22, 30, 35). However, the efficiency of transfection depends on the cell type, as doesthe ability of a given siRNA to silence a particular gene, making interpretation of RNAiexperiments difficult.The use of RNAi in living mice has also been widely reported (2, 12, 18, 20, 21, 28, 29, 31,32, 34), fueling hope that siRNAs may one day be used to treat human diseases. Again, twostrategies for the introduction of RNAi molecules have been used for animal experiments:synthetic siRNA or shRNA delivered directly, or delivery of a plasmid or viral siRNA/shRNAexpression cassette that potentially provides a more stable and long lasting delivery of theRNAi species. Although luciferase reporters were used in a number of these studies (18, 20,21, 32), green fluorescent protein has also proven popular as an alternative reporter (2, 12,18, 28, 31, 34). However, whereas the use of luciferase has allowed quantitative non-invasiveanalysis of gene suppression in live animals, studies using GFP as a reporter have requiredex vivo tissue extraction or cell rescue and FACS to allow visualization of GFP suppression.Moreover, quantification can only be accurately achieved using Northern analysis, whichare time consuming and require sacrifice of the experimental animals. Similarly, detectionof RNAi effects on specific host gene suppression (12, 28, 29) have again required FACS,Northern and western analysis of host tissue and cells.Xenogen’s biophotonic imaging technology provides an ideal strategy to non-invasivelymonitor RNAi in small mammals. In 2002, McCaffrey et al. at Stanford University (20, 21)reported the success of both siRNA and shRNA approaches to reduce luciferase expressionin mice following hydrodynamic transfection methods to introduce the RNAi and luciferase
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